α sma fitc antibody

Anti-Sec23A antibody was kindly provided by Dr. Bin Zhang (Cleveland clinic). Anti-cTAGE5, TANGO1 and Sec12 antibodies were raised and purified as described previously^{16 (link), 18 (link), 19 (link)}. Anti-collagen I antibody (SP1.D8) was obtained from Developmental Studies Hybridoma Bank. Other antibodies were purchased from following companies: α-SMA (Abcam), CREB3L2/BBF2H7 (Atlas Antibodies), Sar1A (Novus Biological), β-actin (Chemicon), GAPDH (Chemicon), α-SMA-FITC antibody (SIGMA), Sar1B (Abnova), Smad2 (Cell signaling), pSmad2 (Cell signaling).

Tissues were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose and crossed into 6 μm sections (LEICA, RM2265). For Elastin Van-Gieson staining, sections were stained with Weigert Solution for 5 min, directly immerged into Differentiation Solution (1% hydrochloric acid alcohol), and then flushed with water. Van-Gieson Dye Solution was used to re-stain the sections for 5–6 min. A microscope (Olympus IX71) was used to observe images.
For immunofluorescent staining, sections or cells were fixed with 4% paraformaldehyde for 15 min, permeabilizated with 0.2% Triton X-100 for 3 min, and incubated with blocking buffer containing 10% goat serum for 1 h at room temperature. Antibody against CD62P (Biolegend, 1:100), Lamtor1 (CST, 1:200), phosphor-p70S6K (Thr389) (CST, 1:200), or p-Src (CST, 1:200) was diluted in blocking buffer and incubated overnight at 4°C. Sections or coverslips were washed with TBS three times and incubated with secondary antibody (Alexa Fluor 568-conjugated goat anti-rabbit IgG, 1:1000, Invitrogen) and α-SMA-FITC antibody (Sigma, 1:500) for 2 h at room temperature. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI) for 10 min. The images were obtained using a confocal laser scanning microscope (Fluoview 1000, Olympus).

Cells were subjected to fixation using 10% formaldehyde followed by cell membrane permeabilization with 0.5% triton X-100. Immunofluorescence staining was performed using DDR2 primary antibody (1:50) (rabbit polyclonal, Santa Cruz Biotechnology, CA, USA) and donkey anti-rabbit-TRITC secondary antibody (1:1000) (Abcam, MA, USA); α-SMA-FITC antibody (1:100) (Sigma Aldrich, St. Louis, USA); 4′-6-diamidino-2-phenylindole (DAPI) (Southern Biotechnology Associates, Birmingham, Alabama). The images were captured by a digital camera connected to an inverted fluorescence microscope (Olympus IX81, Tokyo, Japan). Image overlay was performed using ImagePro Plus 6.0 (Media cybernetics. Inc., Bethesda, MD).